The present invention relates to an improved method for direct detection of analytes, where sensitivity of color changes in liposomes which occur in response to selective binding of analytes to their surface is improved by doping with promoter molecules.
CONVENTIONAL ASSAY METHODS
Analytical Chemistry Analytical chemistry techniques have been used for many years to determine such medical parameters as hematocrit levels. While useful in their own right, analytical chemistry methods are of limited or no practical applicability to many biological systems in which assessment would be valuable. Unless expensive and cumbersome gas chromatography methods are used, large quantities of analytes are generally required to accomplish such methods. Often, quantitative results are limited or not available. However, such techniques are used for such basic chemical tests as creatinine assays.
Microbiology and Pathology Methods Another approach to medical-biological systems analysis has been direct microscopic observation using various cell-staining and classic pathology techniques. Augmenting these capabilities have been well developed microbiological techniques, such as culturing, colony characterization, and observation of metabolic and nutrient limitations. Most of medical science have been developed using this basic arsenal of analytic techniques. While culturing and direct tissue observation techniques have served as the bulwark of medical detection processes for many years, they have important limitations.
Pathological analysis of patient tissues to determine the development of a disease state and the identification of the causative pathogen generally requires an invasive procedure. Culturing a pathogen from various body fluid or other samples is typically time consuming and expensive.
Immunoassays A breakthrough in medicine occurred with the development of immunoassay techniques. In these methods, an antibody is developed which will specifically bind to a target of interest. While costly in both their development and production, antibodies from animals allowed a very accurate analysis of a number of analytes which had previously been virtually unassessable in both research and particularly clinical situations.
An important technical advancement in immunoassay was the development of monoclonal antibodies. Instead of exposing an animal to an analyte and harvesting its full range of antibodies, in this technique a single spleen cell of a sensitized animal is rendered immortal and cloned, providing multiple identical copies. The resulting cell line is then cultured to produce a very specific and pure antibody product.
In immunoassay systems the antibody can be labeled in some way so that the binding event can be detected. This can be done with a dye, fluorescent, radioactive or other label. Conversely, if binding inhibition occurs between a known amount of introduced, labeled analyte and the material to be analyzed, the diminution of the signal will indicate the presence of test analyte. If the agglutination of the antibody particles is of sufficient volume and density, the formation of a precipitant can also serve to signal the presence of an analyte.
In recent years, the research and medical communities have come to rely heavily on immunoassay techniques to detect and quantify biological materials. While successful in many respects, the indirect nature of immunoassay methods as well as their dependence on antibody materials results in a variety of complications, problems, and assay limitations. The development and production of antibodies remain expensive, and these molecules are sensitive to environmental changes. Also, only those materials to which antibodies can be produced can be detected by these systems.
LANGMUIR-BLODGETT FILM ASSAYS
The techniques of molecular self-assembly, such as that described by Swalen et al., (Lanqmuir, vol. 3, page 932, 1987) as well as Langmuir-Blodgett (LB) deposition (Roberts, Ed. Lanqmuir-Blodqett Films, Wiley, New York, 1966) have been used for coating surfaces with a well-defined, quasi two-dimensional array of molecules. The initial use for this new advancement was for materials science applications such as wetting (Whitesides, et al., Lanqmuir, vol. 6, p. 87, 1990) and friction (Novotny et al., Lanqmuir vol. 5, p. 485, 1989).
These bilayer films are also used as immobilizing supports for analytic reactions. Biosensors based on LB films can detect molecules of diagnostic significance such as glucose (Okahata, et al., Thin Solid Films, vol. 180, p. 65, 1989) and urea (Arisawa, et al., Thin Solid Films, vol. 210, p. 443, 1992). In these cases, classic analytical chemistry systems are immobilized on the films in order to improve the readout of the test results and otherwise simplify and improve the detection capabilities of the test procedure.
The detection of receptor-ligand interaction is generally accomplished by indirect assays such as the enzyme-linked immunosorbent and radio-labeled ligand assay. Although biotechnological functionalized films have led to elegant examples of molecular recognition at an interface, the problem of transducing the molecule recognition event into a measurable signal has remained a difficulty until the advent of the subject invention.
In the case of biosensor devices, detection is generally carried out by coupling the LB films to a secondary device such as an optical fiber (Beswick, Journal Colloid Interface Science, vol. 124, p. 146, 1988), quartz oscillator (Furuki et al., Thin Solid Films. vol. 210, p. 471, 1992), or electrode surfaces (Miyasaka, et al., Chemical Letters, p.627, 1990).
Some of the analyte bound films provide for fluorescent label, where the fluorescence or its quenched state indicate the occurrence of a binding event (Beswick, Journal Colloid Interface Science, vol. 124, p. 146, 1988). In some cases, these detection materials have been embedded in the surface of the supporting bi-lipid layer (Tieke, Advanced Materials. vol. 3, p. 532, 1991).
Polydiacetylene films are known to change color from blue to red with an increase in temperature or changes in pH due to conformational changes in the conjugated backbone (Mino, et al., Lanqmuir, vol. 8, p. 594, 1992; Chance, et al., Journal of Chemistry and Physics, vol. 71, p. 206, 1979; Shibutag, Thin Solid Films, vol. 179, p. 433, 1989; Kaneko, et al., Thin Solid Films, vol. 210, p. 548, 1992).
FUNCTIONALIZED LIPOSOMES
Unpolymerized liposomes expressing sialic acid residues have been extensively used as model systems to study the interaction between influenza virus and cell surfaces (Ott, et al., European Journal of Pharmacological Science, vol. 6, p 333, 1994). These liposomes are typically made of such lipid materials as cholesterol and egg phosphatidylcholine (Kingery-Wood, et al, Journal of the American Chemical Society, vol. 114, p 7303, 1992).
A therapeutic functionalized liposome which is produced through polymerization was developed by some of the inventors (Spevak et al, JACS, p 115, 1993). The standard in the field is to progress with the polymerization procedure until the materials are fully red, indicating that the polymerization is complete. This was the procedure used in the above cited publication.
While it has been a goal of the research community to exploit this characteristic in the detection of binding events, researchers have yet to develop a method using this phenomenon in practical applications.